Role of starvation in detachment of Pseudomonas aeruginosa biofilms
by Baochuan Huang
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Chemical Engineering
Montana State University
© Copyright by Baochuan Huang (2000)
Abstract:
Biofilms of Pseudomonas aeruginosa grown in continuous flow reactors spontaneously detached after
the flow was stopped and the biofilm stood in a static aqueous environment for three days. The mean
areal viable cell density was 9.2 log cfu cm^-2 before stopping flow and 7.9 log cfu cm^-2 after the
static period. Similarly, a 1.2 log reduction in areal total cell density was measured between the same
time points. The biofilm matrix appeared to progressively dissolve during the static period, as judged
visually. Treatment of the biofilm with 5% formaldehdye immediately prior to stopping flow prevented
detachment. Treatment with 200 mg/L chloramphenicol, a protein inhibitor, did not prevent
detachment. When, instead of stopping medium flow, the flow was switched to a medium lacking
carbon, the same extent of detachment still occurred. In static experiments in which concentrated
nutrients were periodically amended to the reactor, detachment was largely inhibited. These results
point to a role for carbon starvation in triggering the detachment process. Role of Starvation in Detachment of
Pseudomonas aeruginosa Biofilms
by
Baochuan Huang
A thesis submitted in partial fulfillment
of the requirements for the degree
of
Master of Science
in
Chemical Engineering
MONTANA STATE UNIVERSITY
Bozeman, Montana
December 2000
APPROVAL
of a thesis submitted by
Baochuan Huang
This thesis has been read by each member of the committee and has been found to
be satisfactory regarding content, English usage, format, citations, bibliographic style,
and consistency, and is ready for submission to the College of Graduate Studies.
PLl a
Dr. Phil S. Stewart
Loi/. 2 9 Qjdov
(Signature)
Date
Approved for the Department of Chemical Engineering
Dr. John Sears
(Signature)
Approved for the College of Graduate Studies
Dr. Bruce R. McLeod,
(Signature)
Date
Ill
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a master
degree at Montana State University, I agree that the library shall make it available to
borrowers under rules of the library.
IfI have indicated my intension to copyright this thesis by including a copyright
notice page, copying is allowable only for scholarly purposes, consistent with “fair use”
as prescribed in the U. S. Copyright Law. Request for permission for extended quotation
from or reproduction of this thesis in whole or in parts may be granted only by the
copyright holder.
iv
ACKNOWLEDGMENTS
First I want to thank Dr. Phil Stewart, my advisor, for his guidance and
encouragement through all my unforgettable days, at this university. Without this help it
would be impossible for me to finish this research. I also want to thank Dr. John Sears
for his important guidance in my academic development and Dr. James Duffy, for the
active role he plays in my committee. Thanks to all the members in Center for Biofilm
Engineering. They have made my experience here a sweet memory.
My wife, Lei Wang, has been supporting me all the time. She doubles my
happiness and shares my sadness. I especially acknowledge my parents, Shichu Huang
and Minghui Lei, for their love and endless care, over all the waves of the Pacific Ocean.
TABLE OF CONTENTS
1. INTRODUCTION....................
I
Pseudomonas aeruginosa biofilm............................................................................ \
BiofIlm detachment.................................................................................................. 2
Enzymes that may cause biofilm to detach.............................................................. 4
Thesis goal............................................................................. .................................. 6
2. MATERIALS AND METHODS............................................................................ 7
Microorganism and media........................................................................................ 7
Reactor system and operation. ...........................................................; .................... 9
Reactor sterilization................................................................................................ 11
Stainless steel (316L) slides pre-treatment............................................................. 11
Biofilm culture procedure.............................. ....................................................... 11
Detaching process and sampling............................................................................. 12
Total cell count............................. .............. ..............;........................................... 14
Living cell count..:....................................................... ......................................... 15
Total organic carbon assay..................................................................................... 15
Cryosectioning and staining of biofilm........................................................ .
16
3. RESULTS.......................:....................................................................................... 17
Static biofilm detachment...................................... .............................. .................. 17
Time Scale of biofilm static detachment......................... ........................................20
Biofilm detachment under both continuous shear and starvation............. '............. 22
Effect of some antimicrobial agents on biofilm detachment.................................... 23
Effect of nutrient amendment on biofilm detachment.............................................. 27
Photo of cells shed from Biofilm.............................................................................. 34
4. DISCUSSION...............................
36
Evidence of static biofilm detachment...................................................... ..............36
Universality of biofilm static detachment................................................................ 38
Influence of antimicrobial agents..............................................................................38
Role of starvation..................................................................................... i.............. 40
Passive model............................................................................................................ 41
Cell-cell signaling model........................................................................................... 42
Physiological change of bacteria during starvation................................................... 44
vi
5. CONCLUSION............................
45
6. RECOMMENDATIONS FOR FUTURE WORK.................................................... 46
REFERENCES.............................................................................................
43
APPENDICES............................................................................................................... 53
vii
LIST OF TABLES
Table
-
Page
1. Composition of glucose minimum medium.............................................. 7
2. Composition of trace element solution......................................................8
3. Areal cell numbers of biofilm before and after detachment
in a static aqueous environment for three days......................................18
4. TOC and cell numbers in biofilm and planktonic phases before
biofilm detachment............................................................
5. TOC and cell numbers in biofilm and planktonic phases after
biofilm detachment................................................................................. 19
C
-
6. Areal cells remaining in the biofilm after detachment in a flowing
environment under starvation conditions................................................ 22
7. Areal total cells and living cells remaining at the substratum after
static detachment in the presence of antimicrobial chemicals................ 27
19
viii
LIST OF FIGURES
Figure
. '
Page
1. Molecular structure of alginate...................................................................... 5
2. Drip flow reactor...................................................... .....................................9
3. Drip flow reactor biofilm-culturing system.................................................. 10
4. Static biofilm detachment in drip flow reactor.............................................13
5. Locations of twenty microscopic fields on the membrane for
total cell count............................................................................................ 15
6. Biofilm detachment as a function of time...................................................... 21
7. The influence of some chemicals on the growth of PA Ol............................ 24
8. Effect of antimicrobial chemicals on biofilm detachment............................ 26
9. Effect of nutrient amendment on static biofilm detachment...........................28
10. Combined effect of nutrient amendment and chloramphenicol on
biofilm detachment........................................................:............................ 29
11. Combined effect of nutrient amendment and chloramphenicol on
living cells in planktonic phase and biofilm phase......................................30
12. Frozen cross section of Pseudomonas aeruginosa biofilm picture
before detachment...................................................................................... 32
13. Frozen cross section of Pseudomonas aeruginosa biofilm after
three days of static detachment...... ........................................................... 33
14: Image of cells detached from biofilm.......................................................... 35
V
ABSTRACT
Biofilms of Pseudomonas aeruginosa grown in continuous flow reactors
spontaneously detached after the flow was stopped and the biofilm stood in a static
aqueous environment for three days. The mean areal viable cell density was 9.2 log cfu
cm'2 before stopping flow and 7.9 log cfu cm"2 after the static period. Similarly, a 1.2 log
reduction in areal total cell density was measured between the same time points. The
biofilm matrix appeared to progressively dissolve during the static period, as judged
visually. Treatment of the biofilm with 5% formaldehdye immediately prior to stopping
flow prevented detachment. Treatment with 200 mg/L chloramphenicol, a protein
inhibitor, did not prevent detachment. When, instead of stopping medium flow, the flow
was switched to a medium lacking carbon, the same extent of detachment still occurred.
In static experiments in which concentrated nutrients were periodically amended to the
reactor, detachment was largely inhibited. These results point to a role for carbon
starvation in triggering the detachment process.
I
INTRODUCTION
Pseudomonas aeruginosa biofilm
Biofilms are microbial aggregates that develop and persist at interfaces in both
natural and engineered aquatic environments. Biofilms are composed of microorganisms
embedded in the extracellular polymers (polysaccharides, glycoproteins, and proteins)
they produce (Christensen, 1990; Costerton 1995). Biofilm bacteria have been shown to
predominate in numbers and in metabolic activity in natural (Geesey et al., 1977),
industrial, and medical (Khoury et al., 1992) ecosystems.
Pseudomonas aeruginosa is one important organism that can develop biofilms in
natural settings, industrial systems, and even in the human body. Some strains of P.
aeruginosa can produce large quantities of alginate and therefore have distinctive mucoid
colony morphology. These mucoid strains are most commonly isolated from the
respiratory tract infections that accompany the genetic disease, cystic fibrosis (CF)
(Dogget, 1977). Cystic fibrosis is the most prevalent lethal genetic disease among
people of European descent. The strain used for this research work, PAO I, was a non­
mucoid strain.
It has been well established that biofilms are far more resistant to biocides and
antibiotics than their freely suspended counterparts (Costerton et al., 1987; LeChevallier
et al., 1984; Nickel et al., 1985). The physical and biological mechanisms that render
biofilm microorganisms less susceptible have yet to be established. One hypothesis is
that the bacteria and their exopolysaccharide products significantly reduce the penetration
2
of antimicrobial agents ( Nichols et al, 1989). This transfer barrier is reinforced by the
reactions and adsorption that occur between constituents of the biofilm and antimicrobial
agents. Models of antimicrobial agent penetration into biofilm have been established
(Stewart 1994; Stewart et al., 1995) and are supported by experimental data (Chen et al.,
1993; De Beer et al., 1994; Xu et al.,1995). Another hypothesis relates biofilm
susceptibility to the specific growth rate and phase in the division cycle of biofilm cells
(Evans et al., 1991; Evan et al., 1990; Brown et al.,1990; Duguid et al., 1992).
Biocides and antibiotics are the principle weapons to control biofilms (Srinivasan
et al., 1995). However the dead cells of biofilm may still attach to the surface after
treatment. If a clean surface is needed rather than an inactive but possibly still intact
biofilm, biofilm detachment becomes a crucial process. Furthermore, after biofilm cells
detach from a surface and become planktonic cells, they are easier to kill by antimicrobial
agents. Therefore biofilm detachment and biofilm resistance are closely related.
Biofilm detachment
Biofilm detachment refers to the interphase transport of biomass particles from an
attached microbial film to the fluid compartment bathing the film (Stewart, 1993).
Although detachment has not been investigated extensively, literature data from strongly
different systems prove that it is the primary process that balances microbial growth and,
thereby, determines the extent of biofilm accumulation. During the development of
3
Pseudomonas aeruginosa biofilm in a rotating drum reactor, 85%-95% percent newly
formed biomass of biofilm was released into surrounding medium (Tijhuis et al., 1995).
Bryers has distinguished five categories of detachment processes: erosion,
sloughing, human intervention, predator grazing, and abrasion (Bryers,1988). Erosion
and sloughing are more important in research. Erosion refers to the continuous removal
of individual cells or small groups of cells from the surface of the biofilm. Sloughing, in
contrast, is the detachment of relatively large particles of biomass, whose size is
comparable to or greater than biofihn thickness. Sloughing is a discrete and random
process. Abrasion is caused by collision of solid particles with the biofilm
Biofilm detachment was initially believed to result from a combination of internal
biofilm processes and shear and normal forces exerted by moving fluid on the biofilm
(Characklis, W. G. 1981). Several models have been forwarded using empirical
mathematical expressions to describe detachment rate (Stewart, 1993; Chang et al., 1988;
Rittman, 1982). In different biofihn systems the dominant mechanism of detachment
may be different. For biofilms in fluidized reactors, the turbulence and attrition of bed
fluidization appear to be dominant mechanisms (Chang et al., 1991; Nicolella et al.,
1997). Research on an annular biofilm reactor indicated that detachment rate was directly
related to biofihn growth rate and the factors that limit growth rate would also limit
detachment rate (Peyton, B. M. et al., 1993). No significant influence of shear stress on
detachment rate was observed.
Nutrient concentration has a great influence on biofihn detachment. Some
researchers found that cells detached when nutrient was lacking (Marshall, 1988;
4
Delaquis et al., 1989). Anther study showed that specific detachment rate increased when
nutrient was depleted (Sawyer, L. K., 1998). In contrast, James et al. (1995) reported
cells detached under high nutrient conditions, although different carbon sources were
used for high and low nutrient conditions. Peyton et al. (1993) included nutrient factors
as growth rate inhibitor in their detachment model and this growth rate-dependent
detachment model fitted data better than others in their experimental system.
Enzymes that may cause biofilm to detach
Extracellular polymers (polysaccharides, glycoproteins, and proteins) anchor cells
to a surface. Therefore if these polymers are broken down by enzymes, the biofilm will
detach (Aldridge, I. Y. et al, 1994; Brisou, J. F.; Sutherland, I. W., 1995; Wiatr, C..L.
1990). Combining biofilm-degrading enzymes with cell-killing enzymes can remove and
deactivate biofilms (Johansen, C., et al., 1997). Studies also show that the detachment of
Streptococcus mutans biofilm is mediated by an endogeneous surface protein releasing
enzyme (SPRE) activity (Lee, S. F. et al., 1996). SPRE can cause monolayer biofilm
cells to detach under minimal shear stress.
A key component of the extracellular polysaccharides of Pseudomonas
aeruginosa biofilm is alginate. Alginate is a linear random polymer of |3-l-4-linked Dmannuronic acid and L-guluronic acid (Figure I). The mannuronate residues are
modified to various degrees by O-acetyl groups. Alginate has been shown to play an
5
COOCOO-
Figure I. Molecular structure of alginate.
important role in biofilm formation by increasing the adherence of bacteria to the
substratum (Ramphal et al., 1985; Mai et al., 1993). It is also involved in the chronic
bronchopulmonary infections in cystic fibrosis patients by protecting against antibioticmeditated and polymorphonuclear Ieukoycyte - directed phagocytosis and killing ( Bayer
e ta h ,1992).
Pseudomonas aeruginosa itself synthesizes an enzyme that can break down
alginate molecules - alginate lyase. The gene coding this enzyme is algL. This enzyme
has been isolated and characterized (Eftekhar et ah, 1994; Linker et ah, 1984). It has
been found that alginate lyase modifies alginate molecules in alginate synthesis.
Although some bacteria are capable of digesting alginate and using it as carbon source, it
is still unknown if Pseudomonas aeruginosa can do so.
The role of P. aeruginosa alginate lyase in cell sloughing from agar colonies was
investigated ( Boyd, A., et ah, 1994). Results showed that increased expression of the
alginate lyase in mucoid strain 8830 led to alginate degradation and increased cell
sloughing. On the contrary, Davies (1996) found that P. aeruginosa biofilm did not
6
detach even when the alginate lyase activity was elevated unless the permeabilizing
agents 0.3mM NaCl or SDS (sodium dodecyl sulfate) were,present. Adding purified
alginate lyase alone to a cell cluster can not cause it to dissolve (Davies, 1996).
Allison et al. (1998) found that Pseudomonas fluorescens B52 biofilm lost
exopolymers and biomass when subject to starvation. An exopolysaccharide lyase
activity was detected in the media taken from dense biofilm cultures. Another researcher
reported that Pseudomonas aeruginosa biofilm cultured in a flow cell reactor detached
after the medium supply was stopped and the biofilm stayed in the static environment for
73 hours (Davies, 1996). It is reasonable to assume that a starvation environment
prevailed in this system.
Cell-cell communication signals, such as homoserine lactones, have been found to
play an important role in biofilm formation (Davies, 1998). Some research seemed to
indicate involvement of signaling in biofilm detachment (Allison, 1998) as well. The
addition of iV-acyl-hexanoyl homoserine lactone to the medium appeared to expedite
detachment. One hypothesis is that the signaling molecule may regulate the activity of
some degrading enzymes, which cause biofilm to detach.
Thesis goal
The goal of this thesis was to investigate the effect of starvation on biofilm
detachment in a static aquatic environment.
7
MATERIALS AND METHODS
Microorganism and media
The bacterium used in this study was Pseudomonas aeruginosa strain PAOL It is
a nonmucoid strain that was isolated from a burned wound.
Glucose minimal medium was used in biofilm growth in two concentrations: Ig/L
Glucose minimal medium and O.lg/L glucose minimal medium (Wentland, 1995).
Glucose in the medium must be added separately through a 0.22 pm filter to avoid
carbonization during autoclaving.
Table I. Composition of glucose minimal medium
Chemicals
Strong (g/L)
Standard (g/L)
Glucose
I
0.1
NH4Cl
0.36
0.036
Na2HPO4
13.632
1.3632
KH2PO4
6.56
0.656
MgSO4-TH2O
0.056
0.011
Trace
Iml
0.1ml
8
Table 2: Composition of trace element stock solution
Chemicals
Concentration (mg/L)
(NH4)6M07024
8.96
ZnSO4-TH2O ■
908.8
MnSO4-TH2O
T2.96
CuSO4-SH2O
IT.92
Na2B4O2-1OH2O
8.96
FeSO4-TH2O
101T.6
(HOCOCH2)3N
1280
Co(NO3)2-SH2O
21.32
I
9
Reactor system and operation
Drip flow reactors were used to cultivate biofilms (Fig. 3). Biofilm grows on the
inclined surface of inoculated stainless steel slides that are bathed in a dropwise flow of
medium. The reactor has four chambers. At the bottom of each chamber resides one
stainless steel slide. Sterile medium is pumped continuously (50 mL/hr) onto the
elevated end of the slide. Since the reactor is positioned on a slope ( 10°), the medium
flows down the slide surface and then out of the reactor through a drain port and into a
waste vessel. The vents on top of the reactor maintain an aerobic environment in the
reactor. It usually took four days to develop a mature biofilm on the slide using O.lg/L
glucose minimal medium. Fig. 2 shows the operation of the reactor schematically.
medium drip flow in
air vent
biofilm
substratum
to waste bottle
Figure 2. Drip flow reactor
10
Nutrient, O2
4
Figure 3. Drip flow reactor biofilm-culturing system
11
Reactor sterilization
Reactors were sterilized by autoclaving: Treated slides (see below) were fixed to
the bottom of the reactor chambers with a small piece of autoclave tape. The reactor lids
were laid on top, blit left unscrewed. The reactor was wrapped with aluminum foil and
autoclaved for 30 minutes. After cooling, all connections were checked to prevent leaks.
Stainless steel (316L) slides pre-treatment
In order to produce steel slides with reproducible surface characteristics, the
)
following treatments were necessary. Slides were first dipped in acetone to remove
grease and allowed to air dry, then transferred to fresh PBS 35 (a surface active agent for
cleaning and radioactive decontamination of lab glassware and instruments) working
solution (ImL PBS in 5OmL BLO) and heated to 50°C for 5 minutes. After being
sonicated for 5 minutes, the slides were rinsed with nanopure water and sonicated again
for 5 minutes. They were rinsed three more times and allowed to air dry. The next step
was to soak the slides in 2.OM HCl solution for two hours, followed by thorough
nanopure water rinse and air drying. The slides were then ready for use in the reactor.
Biofilm culture procedure
A planktonic culture was inoculated with PAOl from an agar plate to a 250mL
flask holding 30 mL of Ig/L glucose minimal medium. This culture was incubated at
35°C for 18 hours with shaking. In a biological hood the effluent tubing of the reactor
12
was clamped off. Fifteen mL of (Xlg/L glucose minimal medium was added to each
chamber of the reactor. One mL of planktonic culture was inoculated to each chamber.
The reactor then stood for 24 hours at room temperature in a horizontal position with no
flow.
After the reactor was connected to a 20 L waste reservoir, the inoculation medium
was drained by unclamping the effluent tubing. The influent tubing was connected to a 20
L carboy containing O.lg/L glucose minimal medium. This medium had been autoclaved
for at least 4 hours. The reactor was inclined on a 10° slope. Sterile needles were
attached to the end of pump tubing and pierced into the rubber caps on the reactor. A
pump (Cole-Parmer Co. Model: 7553-80) fed the medium at a constant rate of 50 mL/hr
to each chamber. Biofilm grew for four days at room temperature, which was 22°C.
Detaching process and sampling
After a biofilm developed the following procedure was implemented to bring
about detachment. The effluent tube was shut off with a clamp and the reactor was laid
flat. Fifteen mL of medium was filled into each chamber of the reactor. Care was taken
to prevent hydraulic shock to the biofilm, which might cause biofilm detachment. This
addition was therefore allowed to flow slowly along the chamber wall. The biofilm was
allowed to detach statically in this aqueous environment for three days (Figure 4).
13
air vent
influent
biofilm
medium filled in
substratum
effluent
Figure 4. Static biofilm detachment in drip flow reactor
A second kind of biofilm detachment experiment was performed under
continuous shear stress. After four days growth the influent tube was switched from
medium to sterile nanopure water. The flow speed remained unchanged. The biofilm
was allowed to detach for three days.
After detachment the medium in the chamber was drained. The reactor was
inclined again and drip-flow continued for 10 minutes at 100 mL/hr to eliminate the
cells floating over the biofilm surface. Each slide was transferred with forceps to a 100
mL beaker holding 20 mL PBS. A small plastic scraper (rubber policeman) was used to
scrape all the biofilm down into the beaker. The sample was homogenized for 30 seconds
using a Ultra-Turrax T25 homegenizer (Janke & Kunkel Co., Staufe i. Br.) and was then
ready for assay of total and living cell counts.
14
Total cell count
A filtration apparatus with chimneys was used to deposit cells on a filter
membrane. The stage of the filtration apparatus was first flushed with filter sterilized
water. A polycarbonate membrane filter was spread on the stage, shiny side facing up. A
chimney was clamped on the stage. The biofilm sample was diluted to the appropriate
order of magnitude cell concentration and ImL of that dilution was dropped slowly and
evenly on top of the membrane. Vacuum was applied to the filter for I minute. The
suction on the filtration apparatus was released. Ten pg/mL DAPI was dropped slowly
and evenly on top of the membrane until it was fully covered (about 0.25 ml ,/filter).
After staining fori 5-20 minutes, the DAPI solution was filtered through. A small drop of
immersion oil was placed on a labeled glass microscope slide. The filter membrane was
put on top of the immersion oil (shiny side up). Another drop of immersion oil was
placed on top of the membrane. A glass cover slip was then placed over the membrane.
Total cell counts were made with epifluorescent microscopy (Olympus BH2RFCA) using a 100X objective and a IOX ocular. Twenty random fields were chosen to
count the cells. The total cells on the membrane were calculated based on the average
number of cells in the twenty fields, the area of one field, and the total area of the
membrane. In order to make 20 fields representative, they should be selected in a
predetermined way (Figure 5).
15
Figure 5. Locations of twenty microscopic fields on the membrane for total cell counts
Living cell count
One mL of the homogenized biofilm sample was utilized to make serial 10 fold
dilutions. The dilution tubes of appropriate magnitude were used to do the living cell
count. A 100 pi sample of the dilution tube was dispensed in IOpl drops onto R2A agar
plates using a dispenser. After overnight 35°C incubation colonies on the agar were
counted. Each colony was taken to represent one colony forming unit (cfu).
Total organic carbon assay
A Dohrmann carbon analyzer (DC-80) was used to measure organic carbon
content of a biofilm sample. The basic reaction involves the oxidation of organic carbon
by KS2O4 to produce CO2, which was quantified by the detector.
16
OC + O2 + KS2O4-------- -— ► CO2 +H2O +RX +RS
The carbon analyzer was first calibrated with a 10 ppm sucrose standard. Since
there were cells and many kinds of long-chain molecules in the biofilm, some tailing
occurred due to insufficient oxidization. This tailing can be minimized by limiting the
TOC concentration in the sample to a low level.
Crvosectioning and staining of biofilm
Biofilm samples were cryoembedded with Tissue-Tek OCT compound (Miles
Inc., Elkhart, IN) as described by Yu et al. (1994). Embedded samples were sectioned
using a Leica CM 1800 cryostat (Leica Inc., Deerfield, IE). The 5pm slices were put on
Superfrost plus microscopic slides (Fisher Scientific Inc., Pittsburgh, PA).
DAPI is a water soluble fluorescent stain that binds to DNA. 200 pi of lOpg/mL
DAPI was dropped on the surface of biofilm section. Staining was performed over a
period of 30 minutes and more DAPI was added as needed to replenish that lost by
evaporation during this time. The remaining DAPI was drained. The slide was allowed to
air dry. Epiftuorescent microscopic pictures were taken with a CCD camera program that
was connected to a microscope (Nikon Eclipse E800) using a 20X objective and a IOX
ocular.
17
RESULTS
This section reports the results of measurements of the influence of environmental
factors on detachment of Pseudomonas aeruginosa biofilm. These measurements include
areal total cell numbers and areal living cell numbers of the biofilm before and after the
detachment as well as biofilm thickness and total organic carbon.
Static biofilm detachment
After three days of static detachment in 0.1 g/L glucose minimal medium, biofilm
detachment was reflected in a decrease in the number of cells on the substratum. Table 3
indicates there was a little more than one log reduction (LR) in biofilm living cells, which
means more than 90% percent of the cells moved into the planktonic phase after the
detachment. The result obtained from living cell counts (LR=Lll) is consistent with that
from total cell counts .(LR=I .17). It was observed visually that the substratum became
visible and the biofilm polymers became increasingly translucent as the static detachment
progressed. In the course of performing total cell counts under microscope, it was also
observed that the proportion of smaller cells increased after detachment.
18
Table 3: Areal cell numbers of biofilm before and after detachment in a static aqueous
environment for three days.
Total cells
Before Detachment
After Detachment
Log Reduction
9.56 ±0.13
8.39 + 0.17
1.17
'
(log(#/cm2))
Living cells
9.15 + 0.20
7.87 + 0.17
1.28
(log(cfu/cm2))
To further confirm this biomass transfer from the biofilm to the planktonic phase,
changes in total organic carbon (TOC) and cell numbers in the two phases were
investigated. TOC comes mainly from the cells and extracellular polymeric substances
(EPS). Results are shown in Table 4 and Table 5, which indicate that the majority of the
biomass (total cell, living cell, TOC) that disappeared from the biofilm phase after
detachment transfered to the planktonic phase. The sum of living cells in the two phases
did not change significantly. The sum of total cells increased by 119%. TOC increased
by 37%.
19
Table 4: TOC and cell numbers in biofilm and planktonic phases before biofilm
detachment.
Before Detachment
Totals
Phase
Biofilm
Planktonic
Logic (living cell)
10.44+0.22
9.08+0.11 ,
10.46
Logic (total cell)
10.64+0.12
9.38+0.02
10.66
TOC (mg)
2.25 ±0.41
0.08 ±0.01
2.33
Table 5: TOC and cell numbers in biofilm and planktonic phase after biofilm detachment.
After Detachment
Totals
Phase
Biofilm
Planktonic
Logic (living cell)
9.56+0.23
10.46+0.21
10.51
Logic (total cell)
9.65+0.17
10.95+0.39
10.97
TOC (mg)
0.55 + 0.17
2.65 ±0.79
3.20
20
Time scale of biofilm static detachment
The extent of detachment in this static situation with time is shown in Figure 6. It
is clear that most of the detachment happened within one day, after which there had been
a log reduction of 0.99 in living cell density. Log reductions in areal living cell numbers
ranged from 1.0 to 1.6 between I and 5 days of detachment time. In order to repeat
experiments, a three-day detach time was chosen for further work.
Average detachment rate coefficients were calculated using the following
equation:
ln(— ) = - k d *t
X (cfu/cm2): cell density after detachment
X q(cfu/cm2): cell density before detachment
h (day"1):
detachment rate coefficient
t (day):
detaching time
For the first 0.5 day period of time, the average detachment rate coefficient was
3.22 day"1. Over the first 3 days, the detachment rate coefficient value was 0.98 day'1.
The detachment rate was much higher in the first 12 hrs than it was over the three day
period.
21
Figure 6. Biofilm detachment as a function of time. Detachment is evaluated as log
reduction in areal living cells.
22
Biofilm detachment under both continuous shear and starvation
Four day old mature biofilms were subjected to glucose starvation by switching
the influent medium to the same medium without glucose. Total starvation was
implemented in other experiments by switching the influent medium to nanopure sterile
water.
Results shown in Table 6 indicate a 1.31 log reduction in total cells after glucose
starvation for 3 days and a 1.37 log reduction after total starvation for three days.
Statistical analysis of these two groups of data revealed no significant difference (P=
0.49). The results from living cell counts were consistent: a 1.02 log reduction for
glucose starvation and a 1.36 log reduction for total starvation (P =0.067).
Table 6: Areal cells remaining in the biofilm after detachment in a flowing environment
under starvation conditions.
No starvation
Total cells
Glucose starvation
9.67 ±0.21
8.36 ±0.10
9.36 ±0.12
8.34 ±0.15
Total starvation
'
8.30 ±0.14
(log(#/cm2))
Living cells
(log(cfu/cm2))
8.00 ±0.09
23
Effect of some antimicrobial agents on biofilm detachment
To investigate the relationship between the physiological state of the cells and the
detachment process, certain antimicrobial agents were applied in this system. These
antimicrobial agents inhibit bacterial growth by different mechanisms. Formaldehyde
can kill the cells and deactivate both the intercellular and cytoplasmic enzymes. Sodium
azide inhibits respiratory activity of the cells. Chloramphenicol binds ribosomes and
stops new protein synthesis. The concentration of each chemical utilized was determined
by its effect on the growth curve of PAOl when added at the early log phase. While 5%
formaldehyde and 200 mg/L chloramphenicol totally stopped cell growth, sodium azide
of 200 mg/L concentration only retarded the growth (Figure 7). Increasing the
concentration of sodium azide to 800 mg/L still did not stop growth completely.
24
O
2
“ ♦ “ control
4
6
Time (hr)
8
10
" "* “ 5% Formaldehyde
"♦~ 2 0 0 m q /L SA_ _ _ _ _ _ _ _ •~i ~“ 200nig/L Chloramphenicol
Figure 7: The influence of some chemicals on the growth of PAO I.
Antimicrobial agents were added at time equals 4 hours.
12
25
The effects of adding these chemicals before static detachment (with the medium)
are shown in Figure 8 and Table 7. While 5% formaldehyde totally stopped subsequent
detachment, addition of 200 mg/L sodium azide had no effect on this process.
Application of 200 mg/L chloramphenicol seemed to reduce biofilm detachment slightly.
These effects were tested for statistical significance (t-test).
26
Control
5%Fomuldelyde
200rng/L SoditiinAzide
200nig/L
Chkiaittpheiucol
Figure 8. Effect of antimicrobial chemicals on biofilm detachment. The Y axis is the
log reduction in areal total cells when certain antimicrobial chemicals were added before
static detachment. Control means no chemical was added.
27
Table 7: Areal total cells and living cells remaining at the substratum after static
detachment in the presence of antimicrobial chemicals. Control means no chemical was
added.
Chemicals
Total cells
• (log (#/cm2))
Living cells
(log (cfu/cm2))
Control (no chemicals)
8.39 ±0.16
8.32 ±0.06
5% Formaldehyde
9.34 ±0.13
4.78 ±0.19
200 mg/L Sodium azide
8.20 ±0.12
8.04 ± 0.02
200 mg/L Chloramphenicol
8.64 ±0.11
8.46 ±0.18
The changes in areal living cells were also measured, with results shown in Table
7. Five percent formaldehyde killed more than 99.99% cells in the biofilm. Neither 200
mg/L sodium azide nor 200 mg/L chloramphenicol (one dose) killed biofilm cells
significantly.
Effect of nutrient amendment on biofilm detachment
To further investigate the role of nutrient starvation in the detachment process,
static detachment experiments were performed in which the nutrients were periodically
replenished. This was done by adding a small volume of concentrated medium every 12
hours.
28
CN
<
control
O.lg/L
IgfL,
refreshing
Figure 9. Effect of nutrient amendment on static biofilm detachment. The Y axis
represents areal cell numbers remaining at the substratum after static detachment. The
bars at right show data from experiments in Ig/L glucose minimal medium with periodic
medium amendment. The middle bars show detachment in O.lg/L glucose minimal
medium without medium amendment.
29
6.00E+10
5.00E+10
control
No chloramphenicol
200mg/L
chloramphenicol
Figure 10. Combined effect of nutrient amendment and chloramphenicol on biofilm
detachment. Control represents total cell number before detachment. The middle column
is the total cell number after detachment in amended medium without chloramphenicol.
The right column is the total cell number after detachment in amended medium with 200
mg/L chloramphenicol. Every 12 hours, 1.5 mL medium was taken out, 0.5 mL 10 g/L
glucose minimal medium along with 1.0 ml of 3 g/L chloramphenicol were amended.
30
3.50E+10
ng cell number (cfu
3.00E+10
control
No chloramphenicol
200mg/L
chloramphenicol
Figure 11. Combined effect of nutrient amendment and chloramphenicol on living cells
in planktonic phase and biofilm phase. Control represents living cell number before
detachment. The middle column is the living cell number after detachment in amended
medium without chloramphenicol. The right column is the living cell number after
detachment in amended medium with 200 mg/L chloramphenicol. Every 12 hours, 1.5
mL medium was taken out, 0.5 mL 10 g/L glucose minimal medium along with 1.0 ml of
3 g/L chloramphenicol were amended.
31
The data in Figure 9 indicates that nutrient addition stopped detachment from
occurring. A t-test showed that the total cell numbers in the control and nutrient amended
experiment were statistically significantly different (p < 0.001). No statistical difference
was obtained between the number of areal biofilm cells after detachment in replenished
Ig/L glucose minimal medium and the number of cells before detachment (p = 0.11).
Figure 10 shows that biofilm detachment was mostly stopped when both nutrient
addition and 200 mg/L chloramphenicol were applied in the system. There was no cell
growth in both phases, as indicated by Figure 11. About 90% of the living cells were
killed.
Biofilm detachment was visualized by microscopic examination of frozen cross
sections (Figure 12, 13). The change in biofilm thickness after three days of static
detachment was determined by image analysis. Before detachment the average thickness
of the biofilm was 334 pm; after three days of static detachment the average thickness
had decreased to 27 pm. This is consistent with cell count data showing that over 90% of
the cells detached from the surface.
Figure 12. Frozen cross section of Pseudomonas aeruginosa biofilm picture before
detachment
33
Figure 13. Frozen cross section of Pseudomonas aeruginosa biofilm after three days of
static detachment
34
Photo of cells shed from biofilm
To investigate whether the cells detached from biofilm individually or in clusters,
microscopic images of the cells that detached from biofilm were taken. Since the cell
density was too high, serial dilution of the suspension (to IO3) was made before it was
filtered onto a membrane and stained with DAPI. Figure 14 shows that most of the
detached cells were individual cells. However, a few small agglomerates were directly
observed in the medium after detachment. Due to dilution they did not appear on the
membrane.
35
Figure 14. Image of cells detached from biofilm.
36
DISCUSSION
This chapter summarizes the evidences of static biofilm detachment, discusses the.
influences of antimicrobial agents on biofilm detachment, analyzes the role of starvation
in biofilm detachment. Two models of static biofilm detachment are then put forward
and discussed based on the data in this research.
Evidence of static biofilm detachment
The biofilm detachment phenomenon in this research was visible to the naked
eye. Before detachment the substratum was covered with chunks of biomass and loosely
connected polymer. During detachment, the polymers became progressively more
translucent. After detachment the substratum became visible and the polymer
disappeared. This observation naturally led to the hypothesis that the polymer was
degraded and the biofilm cells were released to the surrounding medium and turned into
planktonic cells.
To further confirm and quantify this detachment, several experiments were
performed. Ninety percent of the cells in the biofilm were released to the surrounding
planktonic phase after three days static incubation. The sum of the number of living cells
in two phases remained unchanged in these experiments. However, the sum of the
number of total cells in the system increased by 119%. This increase could have been
37
caused by the growth of planktonic cells, and possibly biofilm cells, due to the
availability of dissolved carbon source at the beginning of the experiments. If the
number of cells that die is of same order of magnitude as the number of new cells produced by growth, the number of living cells in the system will not change very much.
To determine how many new cells can grow out of O.lg/L glucose minimal medium, a
control experiment was performed, in which the reactor holding 15 mL O.lg/L glucose
minimal medium was inoculated with a small amount of bacteria. There was no biofilm
in the reactor. After three days incubation at 18°C, the average cell density reached
1.83E+08 cfu/mL. The calculated yield coefficient, Yx/s (number of cells/g glucose),
was 1.83E+12. According to this the maximum planktonic growth in the reactor is
2.75E+9 cfu after three days, which is only 10% of the total planktonic cells ( 2.88E+10
cfu) after detachment. This calculation reveals that most of the planktonic cells in the
reactor after three days come from biofilm detachment. The actual planktonic growth in
the reactor should be much less than 2.75E+9 cfu because the majority of the carbon
source will be used by the high-density biofilm cells. The increase in TOC (by 37%)
could be attributed to systematic experimental error. Large molecules such as alginate
can not be oxidized completely, so the TOC value measured could be lower than the true
value. Iflarge molecules are hydrolyzed to smaller ones after detachment, the apparent
total TOC would be expected to increase after detachment, just as was observed.
Frozen cross section pictures of the biofilm directly reveal a ninety percent
decrease in biofilm thickness after detachment. This is consistent with results of total and
living cell counts.
38
In summary, a P. aeruginosa biofilm incubated under static conditions for three
days experienced significant biofilm detachment. This phenomenon was demonstrated
by consistent measurements of total cell numbers, viable cell numbers, total organic
carbon, biofilm thickness, along with visual observations.
Universality of biofilm static detachment
Although several researchers have reported static biofilm detachment in different
experimental systems, it should be pointed out that it is not a universal phenomenon. In
this research when 1/50 LB broth medium is used instead of 0.1g/L glucose minimal .
medium to culture biofilm, biofilm did not detach under same conditions for as long as
seven days. Biofilm cultured with 1/50 LB broth appeared to be more compact than
biofilm cultured with 0.1 g/L glucose minimal medium. Also, when another
Pseudomonas aeruginosa strain- ERC was used instead of PAOI, the biofilm did not
detach. It seems that the structure of biofilm and the components of biofilm polymers are
also very important factors.
Influence of antimicrobial agents
Five percent formaldehyde (final concentration) completely inhibited the
detachment. The mechanism by which formaldehyde affects biological molecules is
quite complex. It kills cells, denatures enzymes, and even creates bonds among cells and
polymers. Formaldehyde treatment appears to kill 90% of the bacteria (Table 7), thereby
blocking induction of an active detachment process. Formaldehyde might simply have
39
denatured the already present matrix-degrading enzymes, preventing their slow but
continued action on the biofilm. Alternatively, formaldehyde could have crosslinked the
matrix components making them resistant to subsequent degradation. Therefore it is hard
to reach a specific conclusion out of this. But the formaldehyde result does suggest that
static biofilm detachment is not simply a pure physical process. It is a biological process
that can be inhibited.
The appliance of 200 mg/L sodium azide did not affect the detachment. Since
200 mg/L sodium azide only moderately inhibited planktonic growth of Pseudomonas
aeruginosa cells, we hesitate to conclude that respiratory activity of the cells is not
needed for detachment.
A parallel experiment with 200 mg/L chloramphenicol led to statistically different
detachment (p = 0.043) as compared with control. Chloramphenicol is an inhibitor of
protein synthesis. This may suggest that stopping new enzyme synthesis reduced
detachment to a small extent (20% or so). But this also shows that majority of the
detachment was accomplished by enzymes already available at the beginning of
detachment. The effect of 200 mg/L chloramphenicol on planktonic Pseudomonas
aeruginosa is significant. It completely stopped the growth of planktonic cells.
The concentrations of antimicrobial agents were determined by planktonic
experiments. Their effects on biofilm cells may be lessened due to the well-known
increased resistance of biofilm. This should not be ignored in considering the results. If
the physiological state of biofilm cells is less influenced by the antimicrobial agents as
compared with planktonic cells, the effect of these antimicrobial agents on detachment
40
could be underestimated. While 5% percent formaldehyde killed more than 99.99% of
biofilm cells, neither 200 mg/L sodium azide nor 200 mg/L chloramphenicol (one dose)
killed biofilm cells significantly. We may need further evidence that they do have
physiological influence on biofilm cells.
Role of starvation
The static detachment happened in O.lg/L glucose minimal medium. Since the
areal cell density of the biofilm was greater than IO9cells/cm2, it is reasonable to assume
that the nutrients were depleted within hours. Therefore this detachment happened in a
starvation environment. Did starvation trigger detachment? To answer this question we
designed the following experiment. I g/L glucose minimal medium was used for
detachment with periodic replenishment to prevent starvation. With all other conditions
remaining the same, the usual detachment did not occur. This is strong evidence that
starvation plays an important role in biofilm detachment. However, there is another
question behind this experiment. Since the concentrated medium was amended
periodically, how can we know if the new cell growth in biofilm is playing an important
role here? Chloramphenicol was then used together with amended medium to control
cell growth in the system. Experimental results showed clearly that detachment will not
happen if the carbon source is available, if cell growth is completely stopped.
This conclusion is supported by a second type of experiment, in which biofilm
detached to the same extent when there was a continuous flow of nanopure water for
three days instead of static environment.
41
Passive model
In this subsection, I present and discuss a model of biofilm detachment that does
not require new respiration, protein synthesis, or growth of bacteria to cause detachment.
This is therefore termed a passive model of detachment.
It is known that Pseudomonas aeruginosa produces alginate lyase, an enzyme that
plays an important role in alginate synthesis by modifying alginate molecules. This
enzyme is'surely capable of chopping the intercellular alginate into smaller molecules.
Since EPS are highly complex polymers, there should exist other hydrolytic enzymes that
can degrade them. If some of these enzymes resides on the cell membrane or is released
into surrounding medium, it will continuously hydrolyze EPS in the extracellular space.
If a carbon source is available, newly synthesized polymers can replenish the polymer,
digested and thereby biofilm structure is maintained. However, if the carbon source is
shut off, the extant EPS will be continuously digested into smaller molecules by these
degradative enzymes. When the molecular length of polymers is not long enoygh to keep
cells together, detachment happens.
The results from this project seem to support this model. In the chloramphenicol
experiment, biofilm detachment happened even when protein synthesis was blocked. To
rationalize the result of the formaldehyde experiment, in which detachment was
prevented, we hypothesize that 5% formaldehyde denatured degradative enzymes. In
medium amendment experiments the replenishment of concentrated medium stopped the
detachment. The passive model can explain these phenomena well. Whether static
detachment happens or not depends on the amount and status (molecular length and
i
42
structure) of EPS, which is determined by the amount of newly synthesized EPS. If
carbon source is available, biofilm structure is maintained because newly synthesized
EPS can replenish the lost polymer. Otherwise static detachment happens.
One piece of evidence from the literature is consistent with this model. Alginate
lyase activity was detected at low level in a flow cell biofilm culture during the
development of static detachment (Davies, D., 1995).
Cell-cell signaling model
This subsection discusses a model of biofilm detachment that depends on an
active biological induction.
The assumption for this model is: There is some signaling molecule in the
biofilm that triggers the elevated synthesis of alginate lyase or the release of this enzyme
into the medium when its concentration accumulates to certain level. The environmental
factors that may elevate the concentration of a signaling molecule include starvation and
cell density (quorum sensing).
David Davies (presentation, 1999) discussed this model in detail and suggested
that a homoserine lactone acted as the signaling molecule. Although some experiments
(Allison, D. G., 1998) indicated a weak relationship between homoserine lactone and
biofilm detachment, there is still no evidence that detachment happens once homoserine
lactone concentration reaches a certain level.
The experimental results from this project do not support the cell-cell signaling
model of biofilm detachment.
43
First, biofilm detached to the same extent in both static system and flow system
when a starvation environment was created. In the flow detaching system the signaling
molecule is not likely to accumulate because, the flow of nanopure water may
continuously convey the signaling molecule out of the reactor. However, no conclusive
statement is made here since little is known about the mass transfer of signaling molecule
in biofilm.
Another weak point in this comparison should not be ignored either. While there
is no shear stress in static detachment, shear does exist for flowing detachment. Shear
strain may play a role in detachment.
Following experimental design might solve the second problem. Instead of
flowing nanopure water onto the slope of coupons, the reactor could be laid flat. A small
ring would be placed over the outlet of the reactor so that the reactor will hold 15 mL
medium before overflow takes place. Then nanopure water would be pumped into the
reactor. The flow speed is slow (50 mL/hr) so that shear is negligible. The starvation
condition is also maintained. The data from this system could give us more convincing
evidence.
Second, the results from nutrient-amended experiments shows that detachment
did not happen in a static environment if the carbon supply was maintained. The
signaling molecule, if there is any, was allowed to accumulate in this system. Since in
each medium amendment (every 12 hrs) 5 mL biofilm culture was first taken out then 5
mL concentrated medium (Ig/L glucose minimal) was filled in, the signaling molecule
would be diluted by 1/3 every 12 hours. This dilution effect would become negligible if
44
we use 0.5 mL 10 g/L glucose minimal medium for each medium amendment and do not
take out the old culture.
Physiological change of bacteria during starvation
When nutrient in the environment is depleted, the physiological states of bacteria
cells change dramatically and turn gradually into stationary phase. Most of these changes
are genetically regulated. It is still unknown how these genetic regulations are related to
biofilm detachment.
An important difference of cells subject to starvation is their smaller size. This
trait may be a passive change reflecting the tendency of cells entering stationary phase to
complete the last round of cell division, but not increase their mass significantly
(Neidhardt, F. C. et al., 1990). This was supported by direct microscopic observation that
the average cell size after static starvation seemed to be smaller than that before
detachment. This decrease in cell size may influence biofilm structure, which ultimately
affects biofilm detachment.
45
CONCLUSIONS
Based on the experiments conducted with P. aeruginosa (PAOl) pure culture
biofilm, the following conclusions are drawn.
1. More than 90 percent of biomass detaches from P. aeruginosa biofilm during
static incubation for 3 days.
2. Starvation is one of the factors affecting biofilm detachment.
3 . ' Biofilm detachment can be blocked by an increased nutrient level.
4. P. aeruginosa biofilm detaches under flowing conditions when subjected to
starvation.
46
RECOMMENDATIONS FOR FUTURE WORK
Future research work may be continued in the following possible directions.
Influence of antimicrobial agents
The continued search for an antimicrobial agent that will inhibit biofilm static
detachment is a good way to understand the detachment mechanism. Work should
continue with new agents. We can then explain this detachment based on the specific
mechanism of that antimicrobial agent. One difficulty is that some antimicrobial agents
themselves cause a biofilm to detach.
Enzyme activity measurement
If we can measure the degradative enzyme activity, such as alginate lyase, in a
biofilm culture, tracking the activity change while detachment develops may give us
useful information. If the activity remains stable during detachment, it may serve as
further evidence for the passive model of detachment. If an increase of enzyme activity
was detected consistently during static detachment, some regulatory mechanism may be
involved. PAOl is a nonmucoid strain of P. aeruginosa. The alginate lyase activity is
lower than that of the mucoid strain. This may cause difficulty in measuring enzyme
activities.
Another alternative way to investigate the role of alginate lyase is to purify this
enzyme and find cations that inhibit its activity significantly. Then such a cation can be
47
added to the detachment system to measure its influence. The potential problem for this
experiment is that these same cations may change the biofilm structure.
Signaling molecules
A simple way to investigate the involvement of a reported signaling molecule in
static detachment is quite straightforward. This chemical can be added at the start of
static detachment to see if it expedites the detachment. Even if it does, evidence should
be given that the chemical does not influence biofilm in other ways before any conclusion
is made.
48
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APPENDICES
(Raw Data)
54
I. Biofilm living cell densities before and after three day detachment
No.
Before
(log(cfu)/cm2)
After
(log(cfu)/cm2)
I
9.28
7.65
2
9.37
7.86
3
8.95
8.12
4
9:01
7.93
5
7.81
2. Biofilm total cell densities before and after three day detachment
No.
Before
After
(log#/cm2)
(log#/cm2)
I
9.64
8.55
2
9.41
8.39
3
9.64
8.22
55
3. Number of living cells in biofilm phase and planktonic phase before and after three day
detachment
No
Before detachment
After detachment
(log(cfu))
PlanktonicPhase
9.15
I
2
(log(cfu))
BiofilmPhase
10.43 '
9.00
10.46
3
PlanktonicPhase
10.49
BiofilmPhase
9.58
10.42
9.54
10.03
9.11
4. Number of total cells in biofilm phase and planktonic phase before and after three day
detachment
No
Before detachment
After detachment
(log#)
I
2
3
PlanktonicPhase
9.37
9.39
(log#)
BiofilmPhase
10.64
10.46
PlanktonicPhase
10.99
BiofilmPhase
. 9.65
10.91
9.33
10.28
9.43
56
5. TOC of planktonic phase and biofilm phase before and after detachment
;
No
Before detachment
After detachment
(mg)
(mg)
I
PlanktonicPhase
0.084
BiofilmPhase
1.84
2
0.083
2.26
1.75
3
0.066
2:66
2.93
PlanktonicPhase
3.26
BiofilmPhase
0.44
0.54
6. Raw data of areal living cell density during three days of static detachment
No. I
Log Reduction
2 ■
3
0
0.12
0.03
0.14
0.5
0.76
0.80
0.54
I
0.89
1.04
2
0.97
1.79
1.19
1.22
1.08
1.55
Time (days)
3
5
.
1.54
57
7. Raw data of optical density measurements of planktonic cells when antimicrobial
agents were added at fourth hour.
control
Time(hrs)
5%Formaldehyde
Sodium Azide
200mg/L
(Optical density)
Chloramphenicol
200mg/L
0
0.003
0.003
0.003
0.003
2
0.018
0.018
0.018
0.018
4
0.078
0.078
0.078
. 0.078
6
0.196
0.065
0.113
0.061
10
0.437
0.063
0.21
0.066
58
8. Total cell density of biofilm before and after static detachment when certain
antimicrobial chemicals were added at beginning
■
before
detachment
No.
Units
after
Formaldehyde
Sodium Azide
detachment
5%
200mg/L
log((#/cm2))
Chloramphenicol
200mg/L
I
9.64
8.55
9.49
8.06
8.74
2
9.41
8.39
9.32
8.29
8.65
3
9.64
8.23
9.14
8.25
8.53
9. Living cell density of biofilm before and after static detachment when certain
antimicrobial chemicals were added at beginning
k
before
detachment
No. Units
after
Formaldehyde
Sodium Azide
detachment
5%
200mg/L
log((cfu/cm))
Chloramphenicol
200mg/L
.1
9.34
8.26
4.99
8.06
8.57
2
9.55
8.31
4.63
8.03
8.31
3
9.27
8.38
4.71
8.03
8.51
I
59
10. Living cell density of biofilm after detachment in different mediums
No
O.lg/L glucose minimal
log((cfu/cm2))
Ig/L glucose minimal, replenishing
log((cfu/cm2))
I
7.93
8.95
2
7.86
8.89
3
8.12
9.1
4
8.31
9.18
10. Total cell density of biofilm after detachment in different mediums
No
O.lg/L glucose minimal
■log((#/cm2))
Ig/L glucose minimal, replenishing
log((#/cm2))
I
8.4
9.4
2
8.55
9.35
3
8.39
9.44
4
8.22
9.49
60
11. Number of total cells in two phases after detachment in amended medium and
200mg/L chloramphenicol.
control
No chloramphenicol
200mg/L
chloramphenicol
NoAphases Biofilm
planktonic
biofilm
planktonic
Biofilm
planktonic
I (loglO(#))
10.64
9.37
10.32
10.43
10.17
9.97
2
10.46
9.39
10.33
10.44
10.27
9.81
3
10.27
average
10.55
total_cells(#)
3.55E+10 2.40E+9
9.38
10.33
10.44
2.11E+10 2.75E+10
10.23
9.89
1.71E+10
7.82E+9
12. Number of living cells in two phases after detachment in amended medium and
200mg/L chloramphenicol.
Control
No chloramphenicol
200mg/L
chloramphenicol
NoAphases Biofilm
planktonic
biofilm
Planktonic Biofilm
planktonic
I (loglO(cfu))
10.43
9.15
9.90
10.24
8.87
9.02
2
10.46
9.00
9.94
10.22
8.97
8.80
3
9.37
average
10.45
9.08
9.92
10.23
9.07
8.91
totalcells(cfu)
2.79E+10
1.19E+9
8.33E+9
1.69E+10
1.17E+9
8.13E+8
61
13. Measures of biofilm thickness before and after detachment
No.
Before detachment
(|am)
After detachment
(Mm)
I
285
25
2
309
44
3
330
28
4
343
29
5
355
22
6
382
13
i
MONTANA
3 1762 10343353 6
I